The present invention relates to a promoter sequence for the p55 tumor necrosis factor receptor (TNF-R), to its preparation and use.
TNF is a multifunctional pro-inflammatory cytokine which affects a wide range of cellular functions. On the one hand, TNF is involved in the protection of the organism, but on the other hand, when over-produced, it can play a major pathogenic role in several diseases. TNF is known to be involved in inflammatory processes and to be a mediator of the damage to tissues in septic shock1, graft-versus-host reactions2 and in rheumatic diseases3.
TNF exerts these effects by binding to two distinct cell surface receptors, which differ in size (about 55 and 75 kDa) and possess structurally dissimilar intracellular domains, suggesting that they signal differently4-11. Almost all cells express TNF receptors (TNF-Rs), yet the amounts and relative proportions of the two receptors vary in different cell types. These variations are in part developmentally controlled; they are related to the phenotype of the cell and its state of differentiation, and in part can be induced transiently by cytokines and immune stimulatory components of pathogens12-22. Studies of the function of the two TNF-Rs indicate that they have different yet interacting activities23-28, and that their activity level may be correlated to the extent of their expression by the cell29-30. These findings imply that the mechanisms which affect the amounts and relative proportion of the two TNF-Rs can have significant influence both on the nature and the extent of the cellular response to TNF and thus constitute important determinants of the physiological as well as pathological manifestations of the funtion of this cytokine.
In order to inhibit the deleterious effects of TNF, ways were sought which would interfere with the binding of TNF to its receptors. Thus neutralizing antibodies to TNF were raised (EP 186 833). Another approach to the inhibition of the action of TNF was the provision of soluble TNF receptors which compete with the cell surface TNF-Rs for the binding of TNF (EP 308 378 and EP 398 327).
Since binding to its receptors is required for TNF in order to exert its action, if less or no cell surface receptors are expressed, it should be possible to decrease or inhibit the deleterious effects of TNF. By the same token, it may be desired in certain cases to augment the beneficial action of TNF and in such a case this could possibly be achieved by increasing the amount of cell surface receptors expressed.
The present invention provides a promoter sequence of the human p55 TNF-R gene which is located upstream of the 5xe2x80x2 end of the gene and is contained within a 976 bp sequence.
The invention in a preferred embodiment provides the NheI-PstI fragment of the full length genomic clone encoding the human p55 TNF-R.
The invention also provides the NheI-EcoRI fragment of the above NheI-PstI fragment.
The invention further provides the BglII-EcoRI fragment of the above NheI-EcoRI fragment.
A promoter sequence according to the present invention may be employed for diagnosing either inherited or acquired mutations in the promoter region which contribute to the pathology of diseases.
In another aspect, the invention provides sequence motifs present in the above described promoter sequences. Such motifs have been shown to bind certain transcription factors which could be necessary for promoter activity and might be involved in regulation of this promoter.
The motifs may be prepared by deletion of the unwanted sequences upstream and/or downstream of the desired motif in the full sequence, i.e., by employing restriction enzymes to cut the full promoter sequence to arrive at the desired motif, and the resulting motif can then be inserted into a vector together with suitable control sequences and other conventional means required in order to obtain a vector which, on insertion into a suitable prokaryotic strain is capable of expression of the desired motif on culturing of the strain.
The motif thus obtained can be used to screen, e.g., a human genomic library or a cDNA library for factors, e.g., transcription factors, binding thereto. Once these factors have been isolated, purified and identified by any conventional means, their inhibition should inhibit TNF-R formation, while their increased production should cause enhanced TNF-R expression thereby leading to the desired effect of modulating TNF function, i.e. inhibition or enhancement of TNF binding to its receptors.
Since the amount of specific transcription factors present in vivo is not unlimited, inhibition of TNF-R expression a n d consequent inhibition of deleterious TNF effects could also be achieved by the expression of a large number of motifs or motif regions. These will compete with promoters containing such motifs or motif regions for binding of the transcription factors. A xe2x80x9cmotif regionxe2x80x9d comprises the motif itself together with sequences flanking it on both sides, or several motifs connected by parts of the whole promoter sequence and flanked on both sides by sequence parts.
The present invention also provides pharmaceutical compositions comprising a sequence motif according to the invention.
In another aspect, the invention provides pharmaceutical compositions comprising a motif region according to the invention.